Destruction of the epithelium of an exocrine gland in the prophylactic and therapeutic treatment of cancer

ABSTRACT

The present invention provides prophylactic and therapeutic methods of treating the ductal epithelium of an exocrine gland, in particular a mammary gland, for disease, in particular cancer. The methods comprise contacting the ductal epithelium of the exocrine gland with an epithelium-destroying gent, preferably by ductal cannulation, so as to realize a prophylactic or therapeutic effect.

[0001] This application claims priority to U.S. patent application Ser.No. 08/510,623, which was filed on Aug. 3, 1995, and has been convertedto U.S. provisional patent application serial number not yet known. Theinvention disclosed in this application was made with government supportunder NIH planning grant P20 CA/ES66205, “Gene-mediated prevention ofCancer,” and grant NIH 1RO1 CA 57993, “Genetic and hormonal factors inmammary carcinogenesis,” awarded by the National Institutes of Health,and with support of the American Cancer Society under grant RD388,“Targeted disruption of breast cells: a novel strategy for cancerprevention.” Therefore, the government has certain rights in thisinvention.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to the use of anepithelium-destroying agent to destroy the epithelium of an exocrinegland, particularly the mammary gland, in the prophylactic andtherapeutic treatment of disease, in particular cancer.

BACKGROUND OF THE INVENTION

[0003] Exocrine glands are glands that release a secretion external toor at the surface of an organ by means of a canal or duct. Examples ofexocrine glands include, among others, the mammary glands, prostate,liver, gall bladder, pancreas, kidneys, sweat glands, and salivaryglands. Cancers of exocrine glands pose a major health problem,frequently resulting in death. Currently, cancers of the breast andprostate are among the leading causes of death among women and men,respectively.

[0004] The mature human breast comprises from six to nine major ducts,which emanate from the nipple, serially branch into ducts and terminatein lobuloalveolar structures (Russo et al., Lab. Invest. 62(3): 244-278(1990)). This branching network of ducts is composed of epithelial cellsin a supporting matrix of connective tissue and endothelial cells.

[0005] Tissues removed from the human female breast during surgery andautopsy have been examined in numerous studies directed to the natureand site of origin of neoplastic growth. Subgross sampling andhistological confirmation have enabled pathological characterization ofentire breasts, leading to the postulation of the existence of fourmajor possible sites of origin of mammary carcinomas, namely ducts,terminal ducts, ductules, and acini (Russo et al., supra). Ductal originis supported by the presence of more extensive epithelialproliferations, which are presumed to be preneoplastic, in surgicallyremoved cancerous breasts as compared to nonmalignant breasts removedduring autopsies (Russo et al., supra).

[0006] With a cumulative lifetime risk of a woman developing breastcancer estimated to be 1 in 9, there is an urgent need to developtherapeutic methods of treatment that are more effective, less invasiveand accompanied by fewer side effects and prophylactic methods oftreatment that are more effective than increased and intensifiedphysical monitoring and far less extreme than radical mastectomy. Inspite of the recent discovery of the heritable breast cancersusceptibility loci, BRCA1 (Miki et al., Science 266: 66-71 (1994)) andBRCA2, and other cancer susceptibility loci, and the increasing abilityof physicians to identify women with elevated breast cancer risk,prophylactic methods are still currently limited to physical monitoringand prophylactic mastectomy.

[0007] In view of the above, it is an object of the present invention toprovide a method of locally treating an exocrine gland prophylacticallyfor disease. It is another object of the present invention to provide amethod of locally treating an exocrine gland, in particular the mammarygland, prophylactically for cancer. Another object of the presentinvention is to provide a method of locally treating an exocrine glandtherapeutically for disease. Yet another object of the present inventionis to provide a method of locally treating a mammary glandtherapeutically for cancer. Still yet another object of the presentinvention is to provide a method of locally treating a mammary glandboth therapeutically and prophylactically for cancer. These and otherobjects and advantages, as well as additional inventive features, willbecome apparent from the detailed description provided herein.

SUMMARY OF THE INVENTION

[0008] The present invention provides prophylactic and therapeuticmethods of treating the ductal epithelium of an exocrine gland, inparticular the mammary gland, for disease, in particular cancer. Themethod comprises contacting the ductal epithelium of the exocrine gland,in particular the mammary gland, with an epithelial-destroying agent.The ductal epithelium is preferably contacted with the agent by ductalcannulation. The epithelium-destroying agent is preferably a vectorcomprising a thymidine kinase gene, which is used in combination withganciclovir (GCV), which can be systemically administered. Anotherpreferred epithelium-destroying agent is a vector comprising ahypoxanthine phosphoribosyl transferase (HPRT) gene, which is used incombination with hypoxanthine aminopterin thymidine (HAT) nucleotide,which can be systemically administered. Also preferred as anepithelium-destroying agent is a vector comprising a gene, which, upontransformation of a cell of the ductal epithelium and expressiontherein, induces apoptosis or death of the transformed cell. A preferredapoptosis-inducing gene is bclxs. Other preferred epithelial-destroyingagents include a cytolytic virus, such as Vaccinia virus, and ethanol.The preceding methods can additionally comprise contacting the ductalepithelium with a cytokine or hematopoietic growth factor, such asGM-CSF. Also provided by the present invention is a method of treatingthe ductal epithelium of a mammary gland both therapeutically andprophylactically for cancer. The combined therapeutic/prophylacticmethod comprises treating the mammary gland therapeutically by surgery,radiation and/or chemotherapy and contacting the ductal epithelium ofthe mammary gland, either concomitantly or subsequently, with anepithelium-destroying agent, which does not specifically targetcancerous cells. The combined therapeutic/prophylactic method canadditionally comprise contacting the ductal epithelium with a cytokineor hematopoietic growth factor, such as GM-CSF.

BRIEF DESCRIPTION OF THE FIGURES

[0009]FIG. 1 is a graph of the ratio of cell no. in the presence of GCVover cell no. in the absence of GCV (Cell No. +GCV/Cell No. −GCV) versusviral multiplicity of infection (viral MOI), which shows the effect ofthe addition of 10 μg/ml GCV on NMU68 and RBA rat tumor cell lines 6 hrsafter transduction with adenoviral-Herpes simplex thymidine kinase(AdHS-tk) at titers of 0, 100, 500 and 1,000 moi. After the cells weremaintained in the presence of GCV for 3 days, they were counted usingtrypan blue exclusion as a measure of cell viability and cell numberswere normalized to the growth of cells in the absence of ganciclovir(GCV).

DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention is based on the observation that theoverwhelming majority of breast cancers arise from epithelial cells,particularly those epithelial cells which line the ducts of the mammarygland and are collectively referred to as the ductal epithelium. Thepresent invention is also based on the exocrine nature of the mammarygland. Given that the mammary gland is an exocrine gland, it was furtherobserved that the central canal or duct could provide a means ofdirectly accessing the ductal epithelium for localized prophylactic andtherapeutic treatment of cancer. Based on these observations, theprophylactic and therapeutic methods of the present invention weredeveloped.

[0011] Prophylactic Method

[0012] The prophylactic method of the present invention is a method oftreating the ductal epithelium of an exocrine gland prophylactically fora disease that affects the ductal epithelium of the exocrine gland. Themethod comprises contacting, preferably by ductal cannulation, theductal epithelium of the exocrine gland with an epithelium-destroyingagent so as to destroy cells of the ductal epithelium affected by thedisease.

[0013] In one embodiment of the prophylactic method of the presentinvention, the ductal epithelium of a mammary gland is treatedprophylactically for cancer so as to inhibit the formation of cancer ofductal epithelial origin. The method comprises contacting, preferably byductal cannulation, the ductal epithelium of the mammary gland with anepithelium-destroying agent. The agent preferably is a vector comprisinga thymidine kinase gene, such as a Herpes simplex thymidine kinase gene,and ganciclovir, a vector comprising a HPRT gene and HAT nucleotide, acytolytic virus, such as a Vaccinia virus, or ethanol. The method canadditionally comprise contacting the ductal epithelium with a cytokineor hematopoietic growth factor, such as GM-CSF.

[0014] In another embodiment of the prophylactic method of the presentinvention, the ductal epithelium of an exocrine gland, such as a mammarygland, is treated prophylactically for cancer so as to inhibit theformation of cancer of ductal epithelial origin. The method comprisescontacting, preferably by ductal cannulation, the ductal epithelium ofthe exocrine gland with an epithelium-destroying agent to destroy lessthan all of the ductal epithelium so as to inhibit the formation ofcancer of ductal epithelial origin. Preferably, up to about 70%, 80%,85%, 90% or 95% of the ductal epithelium is destroyed.

[0015] The epithelium-destroying agent is preferably a vector comprisinga thymidine kinase gene, such as that from Herpes simplex, andganciclovir, which can be brought into contact with the ductalepithelium by any suitable means, preferably by ductal cannulation or bysystemic administration, a vector comprising a HPRT gene and HATnucleotide, which can be brought into contact with the ductal epitheliumby any suitable means, preferably by ductal cannulation or by systemicadministration, a vector comprising a gene, which upon transformation ofa cell of the ductal epithelium and expression therein, inducesapoptosis or death of the transformed cell, such as bclxs, ethanol, or acytolytic virus, such as Vaccinia virus.

[0016] The above method can additionally comprise the administration ofa cytokine or hematopoietic growth factor, such as GM-CSF. The GM-CSFcan be brought into contact with the ductal epithelium of the mammarygland by any suitable means, such as by ductal cannulation of GM-CSF ora vector comprising a gene encoding GM-CSF, in which case the vector canbe the same vector as the one encoding the thymidine kinase, HPRT orapopotosis-inducing gene, or it can be systemically administered.

[0017] This embodiment of the prophylactic method can be used to treatany exocrine gland. However, it is particularly useful in the treatmentof the mammary gland.

[0018] The above-described prophylactic method of treating a mammarygland is particularly useful in treating a mammary gland in a mammal atrisk for developing breast cancer. The mammary gland can becharacterized as one that has never had a tumor, one that had a tumorpreviously but the tumor is no longer detectable due to other priortherapeutic treatment, or one that has an incipient or occult tumor,preneoplasia or ductal hyperplasia. Normally, hyperplasias and incipientand occult tumors are not detectable by means of physical examination orradiography. Accordingly, the prophylactic method will find use in caseswhere there is reason to take some prophylactic measures, such as whenthere are known inherited factors predisposing to cancers, where thereare suspicious lesions present in a breast with the potential fordeveloping into a malignancy, where there has been exposure tocarcinogenic agents in the environment, where age predisposes to acancer, where cancer of another gland, e.g., the mammary gland of thecontralateral breast, suggests a propensity for developing cancer, orwhere there is a fear or suspicion of metastasis.

[0019] Therapeutic Method

[0020] The therapeutic method of the present invention is a method oftreating the ductal epithelium of an exocrine gland therapeutically fora disease that affects the ductal epithelium of the exocrine gland. Themethod comprises contacting, preferably by ductal cannulation, theductal epithelium of the exocrine gland with an epithelium-destroyingagent so as to destroy cells of the ductal epithelium affected by thedisease.

[0021] In one embodiment of the therapeutic method of the presentinvention, the ductal epithelium of a mammary gland is locally treatedtherapeutically for cancer so as to destroy cancerous and noncancerouscells of the ductal epithelium and inhibit the spread of cancer. Themethod comprises contacting, preferably by ductal cannulation, theductal epithelium of the mammary gland with an epithelium-destroyingagent, which need not, and preferably does not, specifically targetcancerous cells. The agent preferably is a vector comprising a thymidinekinase gene, such as a Herpes simplex thymidine kinase gene, andganciclovir, a vector comprising a HPRT gene and HAT nucleotide, acytolytic virus, such as a Vaccinia virus, or ethanol. The method canadditionally comprise contacting the ductal epithelium with a cytokineor hematopoietic growth factor, such as GM-CSF.

[0022] In the therapeutic method, the epithelial-destroying agent shoulddestroy all of the diseased or malignant epithelium. In addition, theductal epithelium immediately surrounding the diseased/malignantepithelium also preferably should be destroyed.

[0023] Combined Therapeutic/Prophylactic Method

[0024] The present invention also provides a method of treating theductal epithelium of a mammary gland both therapeutically andprophylactically for cancer. The method comprises treating the mammarygland therapeutically with any given therapeutic method, such as thosecurrently known and used in the art. Examples of such methods includesurgical removal of the cancerous tissue, radiation therapy andchemotherapy. The method further comprises contacting, eitherconcomitantly with or subsequently to the therapeutic treatment, theductal epithelium of the mammary gland, e.g., by ductal cannulation,with an epithelium-destroying agent, which preferably does notspecifically target cancerous cells, so as to destroy any remainingcancerous cells and noncancerous cells and inhibit the spread of cancer.The epithelium-destroying agent is preferably a vector comprising athymidine kinase gene, such as a Herpes simplex thymidine kinase gene,combined with ganciclovir, a vector comprising a HPRT gene combined withHAT nucleotide, a cytolytic virus, such as a Vaccinia virus, or ethanol.The method can additionally comprise contacting the ductal epitheliumwith a cytokine or hematopoietic growth factor, such as GM-CSF.

[0025] Alternative Embodiments of Prophylactic & Therapeutic Methods

[0026] Although preferred embodiments have been described above, themethods of the present invention can be used to treat the ductalepithelium of any exocrine gland. Examples of exocrine glands, otherthan the mammary gland, which can be treated with the present inventivemethods include, among others, the prostate, liver, gall bladder,pancreas, kidneys, sweat glands, and salivary glands. The methods areespecially useful in the prophylactic and therapeutic treatment of theductal epithelium of mammary glands.

[0027] Similarly, the methods can be used to treat an exocrine gland forany disease that affects the exocrine ductal epithelium. The methods areparticularly useful in the treatment of cancer, including the stages ofhyperplasia, adenoma, carcinoma in situ, and carcinoma, of ductalepithelial origin.

[0028] Any method can be used to destroy the ductal epithelium. It ispreferred, however, that the destruction is limited to the ductalepithelium or a part thereof.

[0029] Any method of contacting the ductal epithelium can be used toeffect local treatment. Preferably, ductal cannulation is used. Althoughany duct or lobule can be cannulated, it is preferred that the centralcanal or duct be cannulated. Ductal cannulation also enables directinjection of a tumor mass, if desired.

[0030] Any epithelium-destroying agent can be used to destroy the ductalepithelium of an exocrine gland. The agent preferably should not destroycells other than cells of the ductal epithelium and preferably shouldnot result in side effects, the adversity of which outweigh the benefitsof destruction of the ductal epithelium. In no event should the methodsbe used to destroy completely the ductal epithelium of an exocrine glandin the prophylactic/therapeutic treatment of a given disease, whereinthe complete destruction of the ductal epithelium, in and of itself,would result in death of the mammal so treated.

[0031] Examples of agents that can be used in the context of theprophylactic and therapeutic methods of the present invention includecytotoxic agents. Any cytotoxic agent known in the art and suitable forcontacting the ductal epithelium of an exocrine gland of a mammal can beused. In addition to ethanol and GCV described above, other examples ofcyotoxic agents and their prodrugs include genistein, okadaic acid,1-β-D-arabinofuranosyl-cytosine, arabinofuranosyl-5-aza-cytosine,cisplatin, carboplatin, actinomycin D, asparaginase,bis-chloro-ethyl-nitroso-urea, bleomycin, chlorambucil,cyclohexyl-chloro-ethyl-nitroso-urea, cytosine arabinoside, daunomycin,etoposide, hydroxyurea, melphalan, mercaptopurine, mitomycin C, nitrogenmustard, procarbazine, teniposide, thioguanine, thiotepa, vincristine,5-fluorouracil, 5-fluorocytosine, adriamycin, cyclophosphamide,methotrexate, vinblastine, doxorubicin, leucovorin, taxol, anti-estrogenagents such as tamoxifen, intracellular antibodies against oncogenes,the flavonol quercetin, Guan-mu-tong extract, retinoids such asfenretinide, nontoxid retinoid analogues such asN-(4-hydroxyphenyl)-retinamide (HPR), and monoterpenes such as limonene,perillyl alcohol and sobrerol. Preferably, the agent is locallyadministered, especially if administration of the agent is accompaniedby toxic side effects. Otherwise, the agent can be administered by anysuitable route, such as systemic administration.

[0032] A cytolytic virus also can be used as an agent. Any cytolyticvirus can be used as long as the organism mounts a rapid immunologicalresponse to it such that the virus cannot cause disease if it escapesthe ductal epithelium. Examples of cytolytic viruses include Vacciniaviruses and Sindbis viruses, which can also be used as vectors.Preferably, a Vaccinia virus is used. Due to lack of mucosal immunity,Vaccinia infectious particles enter and lyse the breast epithelialcells, yet stromal immunity destroys the particles as soon as they leavethe ductile tree of the exocrine gland, thereby preventing cytolysisbeyond the ductal epithelium. The advantages of Vaccinia administrationare that it eliminates the need for high titer virus, the need to inducecell division in the breast, and the need to administer a drug to effectcell death.

[0033] A vector comprising a suicide gene also can be used as an agent,in conjunction with an agent that destroys the ductal epithelium of anexocrine gland. The vector comprising a suicide gene, upontransformation of a cell of the ductal epithelium and expressiontherein, renders the transformed cell sensitive to theepithelium-destroying agent, increases the sensitivity of thetransformed cell to the agent, converts the agent from a prodrug to anactive drug, activates the conversion of the agent from a prodrug to anactive drug, enhances the effect of the agent or, itself, produces aprotein that is cytotoxic. A preferred suicide gene for use in thepresent inventive methods is the one described above, i.e., a thymidinekinase, such as the one from Herpes simplex, which phosphorylates GCV,which, in turn, inhibits DNA replication. Another example of a suicidegene is cytosine deaminase, which is used in conjunction with5-fluorocytosine. If the vector comprising the suicide gene isadministered locally to the ducts, the cytotoxic agent or precursor canbe administered systemically, since only transfected cells will beaffected. In this regard, the bystander effect, i.e., the death ofneighboring uninfected cells, presumably due to transfer of toxicbyproducts through gap junctions between cells in the same compartment,obviates the need for every cell in the ductal epithelium, which is tobe destroyed, to be infected. However, sufficient time must be allowedbetween contacting the ductal epithelium with the suicide gene and theprodrug, for example, to achieve efficient killing of the breastepithelial cells.

[0034] A vector comprising an apoptosis-inducing gene also can be usedas an agent that destroys the ductal epithelium of an exocrine gland(Vaux, Cell 76: 777-779 (1994)). Examples of apoptosis-inducing genesinclude ced genes, myc genes (overexpressed), the bclxs gene, the baxgene, and the bak gene. The apoptosis-inducing gene causes death oftransfected cells, i.e., by inducing programmed cell death. For example,the bclxs gene, bax gene, or bak gene can be used to inhibit bcl-2 orbcl-x_(L), leading to apoptosis. Where necessary, a vector comprising anapoptosis-inducing gene can be used in combination with an agent thatinactivates apoptosis inhibitors such as bcl-z, p35, IAP, NAIP, DAD1 andA20 proteins.

[0035] Suicide and apopotosis genes can be administered by way of aviral vector, such as an adenoviral or retroviral vector. Adenoviralvectors enable the generation of high titer recombinant viruses(10¹¹/ml) and the efficient transduction of postmitotic cells becauseadenoviral DNA exists as an episome in the nucleus (Verma, MolecularMedicine 1:2-3 (1994)).

[0036] According to one preferred embodiment, the gene can be under thetranscriptional regulation of a Rous sarcoma viral promoter.Alternatively, it can be under the control of an epithelialtissue-specific or cell-specific promoter.

[0037] Uptake of recombinant virus can be facilitated by pretreatment orsimultaneous treatment with polybrene or, for example, in the case of aretrovirus, attachment of the functional fragment of an antibody to theviral particle.

[0038] In another embodiment, the apoptosis gene or suicide gene can bepresent in a recombinant microorganism, which will express the gene. Oneparticularly preferred microorganism for this purpose is the bacteriumListeria monocytogenes.

[0039] Other methods known in the art for introduction of raw DNA intocells can be used in the methods of the present invention.Alternatively, liposomes, complexes between polypeptide ligands forreceptors on mammary ductal epithelial cells, including complexes ofantibodies and functional fragments thereof, and plasmids can be used(Mulligan, Science 260: 926-931 (1993)). Epithelial cell-specificpromoters, such as whey acidic protein (wap), can be used to targetexpression of a given gene, e.g., a suicide gene, in ductal epithelialcells. Use can also be made of wild-type tumor suppressor genes, such asp53 or Mcs-1 (rat), homeobox genes expressed in normal cells but not incancerous cells, and the maspin gene.

[0040] Additionally, the ductal epithelium can be contacted with anagent to effect the scavenging of epithelial cells destroyed inaccordance with the present invention, e.g., a cytokine/growth factor.Suitable cytokines/growth factors include GM-CSF, G-CSF, IL-2, IL-4,IL-6, IL-7, hCG, TNF-α, INF-α and INF-γ. Such factors can be contactedwith the ductal epithelium directly or by expression of a vectorcomprising a gene encoding the factor, in which case the vector can bethe same one that comprises a suicide gene, for example. The factorsstimulate a potent, long-lasting, and specific cell immunity, requiringboth CD4 and CD8 cells. The immune response is designed to scavengedestroyed ductal epithelial cells by generating autoimmunity towardsepithelial cell antigens.

[0041] The ductal epithelium is preferably contacted with the agent byintroduction of the agent through the central canal or duct of theexocrine ductal epithelium, such as by ductal cannulation. However, inthe case of the mammary gland, for example, there are 6-9 major ductsthat emanate from the nipple and serially branch into other ducts,terminating in lobulo-alveolar structures (Russo et al. (1990), supra).Accordingly, in some circumstances, such as those in which even morelocalized treatment is necessary or desired, for example, by the choiceof anti-cancer agent, it may be preferable to contact the ductalepithelium of the exocrine gland through one of the other ducts orthrough a lobulo-alveolar structure as opposed to the central canal orduct. In this regard, ductal cannulation enables intratumoral injection.

[0042] The methods of the present invention can be combined with othermethods of prophylactic and therapeutic treatment in addition to thosecited above, such as methods that target destruction of cancer cells,e.g., by targeting of cell-surface markers, receptor ligands, e.g.,ligands to gastrin-releasing peptide-like receptors, tumor-associatedantigens, e.g., the 57 kD cytokeratin or the antigen recognized by themonoclonal antibody GB24, the extracellular matrix glycoproteintamascin, antisense oncogenes such as c-fos, homeobox genes that areexpressed in cancer cells but not normal cells, tumor-infiltratinglymphocytes that express cytokines, RGD-containing peptides andproteins, which are administered following surgery, lipophilicdrug-containing liposomes to which are covalently conjugated monoclonalantibodies for targeting to cancer cells, low fat diet, moderatephysical exercise and hormonal modulation. For prostate cancer,anti-testosterone agents can be used as well as an inhibitor of cellproliferation produced by prostatic stromal cells and C-CAM, anepithelial cell adhesion molecule.

[0043] The following examples are presented to illustrate the presentinvention, not to limit its scope. The examples make use of the ratmammary tumor model, which has been deemed an appropriate experimentalmodel for understanding breast cancer in humans (Sukumar et al.,Mutation Res. 333 (1-2): 37-44 (1995); Russo et al., supra). In fact,90-100% of female rats develop mammary tumors in this model when theyare administered the carcinogen NMU at 55 days of age (Sukumar, CancerCells 4:199-204 (1990)).

EXAMPLES Example 1

[0044] This example demonstrates the successful delivery of virus andother agents into the mammary ductile tree by a single injection throughthe teat.

[0045] ADV/CMV-β-gal (from Dr. William Burns, Johns Hopkins University)is an adenoviral 5 vector constructed with a β-galactosidase genecontrolled by a cytomegaloviral promoter. It was delivered into themammary gland by injection of a viral suspension in 20 μl of 0.2% trypanblue in Tris buffer through the teat of a rat. The nipple was extruded,and the sphincter removed by excising the nipple. In the rat, the muscleprevents fluid from regurgitating into the breast and had to be excisedin order to visualize the ductal opening and administer the agent.Trypan blue was used as a tracking dye to ensure correct delivery to theductile tree. Injection about 30 days postpartum resulted in the mammaryepithelial tree being clearly visible. Ethyl alcohol (70%) was alsosuccessfully delivered by a single injection through the teat.

Example 2

[0046] This example demonstrates that adenovirus can efficientlytransduce human mammary epithelial cells in vitro.

[0047] ADV/CMV-β-gal was used to transduce HBL100 mammary epithelialcells in vitro. The β-gal enzyme in this construct contains a nuclearlocalization signal and results in dense nuclear staining.—HBL100 cells(10²) were plated in 24-well plates, and transduced with virus atvarious doses and stained with X-gal 48 hrs later. Essentially all cellswere infected at a moi=10⁴.

[0048] This experiment also has been performed in human mammary tumorcells MCF-7 (American Type Culture Collection (ATCC), Rockville, Md.),human mammary epithelial cells MCF-10A (ATCC), and two rat mammarycancer cell lines, RBA (from Dr. Leonard Cohen) and 37-2 (from Dr. C.Marcelo Aldaz) with the same results. More efficient adenoviralconstructs have been used, thereby achieving 100% infection at amoi=10³. These experiments demonstrate successful infection by andexpression of adenovirus carrying the lacZ indicator gene, which permitsstaining the cells blue by the expression of the enzyme β-galactosidase.

Example 3

[0049] This example demonstrates that infection with an AdHS-tkconstruct followed by GCV treatment effectively kills mammary tumorcells in vitro.

[0050] RBA and NMU68 are two rat mammary tumor cell lines derived from aDMBA- and a NMU-induced tumor, respectively[DMBA=dimethylbenz[a]anthracene, NMU=N′-nitro N′-methylurea]. Each cellline was plated at a density of 5×10² in 48-well plates (1.1 cm) andallowed to settle overnight. The next morning, they were transduced withAdHS-tk (Chen et al., PNAS(USA) 91: 3054-57 (1994); obtained from S. Wooand E. Aguilar-Cordova, Baylor College of Medicine, Houston, Tex.) attiters of 0, 100, 500, and 1000 moi, and then, 6 hrs later, GCV (10μg/ml) was added to the culture media. The cells were maintained in thepresence of GCV for 3 days and then counted using trypan blue exclusionas a measure of cell viability. The cell numbers were normalized to thegrowth of cells in the absence of GCV. The results are shown in FIG. 1.More than 80% of the cells of each cell line were killed at a moi of10³.

Example 4

[0051] This example demonstrates the prophylactic effect of the methodof the present invention.

[0052] The mammary glands (6 on each side) of six virgin 50 day oldSprague Dawley rats were injected with AdHS-tk on the left side andtrypan blue on the right side or left untreated. One rat remainedcompletely untreated with the virus or GCV and served as a positivecontrol for NMU-induced tumorigenesis. On the day of surgery, rats weregiven an intramuscular injection of 5 μg estradiol valerate and wereanesthetized with an isofluorane/O₂ mixture. The nipples were cannulatedwith a 33 gauge needle. Twenty μl of AdHS-tk diluted in trypan bluecarrier (1 mM MgCl₂, 20 μg/ml polybrene in 10% glycerol, 0.4% trypanblue in saline) at a concentration of 5×10⁷ particles/μl were injectedinto the duct. Carrier control mammary glands received 20 μl of trypanblue carrier alone. An animal was considered treated when at least threeglands were successfully injected with trypan blue and another threeglands were successfully injected with AdHS-tk. The remaining glandswere left untreated. Twelve hrs later, the rats were injected with 125mg/kg body weight GCV twice daily for three days. The rats were thengiven a second intramuscular injection of 5 μg estradiol valerate andintraperitoneal injections of 100 μg/kg body weight GCV once daily forthree days. Five to seven days after GCV treatment, the rats were givenan intravenous injection of NMU dissolved in 0.05% acetic acid (AshStevens, Colo.; 50 mg NMU/kg body weight) and were subsequentlymonitored for general health and the appearance of tumors at weeklyintervals for 8 months. The results were as follows: No. of No. of Total% of Glands Treatment Tumors Glands with Tumor Untreated 5 12 41.7Control Trypan Blue 5 17 29.4 and Ganciclovir No injection; 5 20 25.0Ganciclovir AdHS-tk and  2* 35  5.7 Ganciclovir

[0053] The above results show that the method of the present inventioninhibits the formation of cancer of ductal epithelial origin in this ratmodel, in which NMU induces the formation of mammary tumors in 90-100%of female rats of similar age. Surprisingly and unexpectedly, thisprophylactic effect was achieved without extensive destruction of themammary glands. These data demonstrate that selective destruction ofepithelial cells, e.g., key stem cells, can be sufficient to provideprophylactic protection against carcinogen-induced tumor formation inthe ductal epithelium of the mammary gland.

Example 5

[0054] This example demonstrates the efficient transfection of mammaryepithelial cells in vivo.

[0055] Lytic Vaccinia virus (10⁶ Vaccinia-HA, which also carries lacZ,in 20 μl 0.2% trypan blue) was injected into the mammary glands throughthe teat of 45 day old virgin rats. Contralateral control glands wereinjected with 0.2% trypan blue. The glands were excised after 3 days.Frozen mammary gland sections were stained with X-gal and counterstainedwith eosin. When Vaccinia-HA was injected via the rat teat, it was ableto infect the epithelial cells. At 3 days post-infection, the X-galstaining was confined primarily to the epithelial cells.

Example 6

[0056] This example demonstrates the cytotoxicity of Vaccinia/HA onHBL100 cells in vitro.

[0057] HBL100 cells (from ATCC, Rockville, Md.) were plated in DME:F12medium (50% Dulbecco's Modified Eagles Medium: 50% Ham's F12 Supplement)containing 10% fetal bovine serum and 10 μg/ml insulin at a density of5×10⁴ cells/well and incubated at 37° C. overnight. Vaccinia/HA, atconcentrations of 0 moi, 0.1 moi, or 1.0 moi, was added to the culturemedium, and the cells were incubated at 37° C. for at least 3 days. Morethan 90% of the cells were dead within 72 hrs of infection at 0.1 moi.

Example 7

[0058] This example shows the death of rat mammary tumor cells inculture by infection with Vaccinia/HA.

[0059] Cells of rat mammary cancer cell line RBA were plated in growthmedium at a density of 5×10⁴ cells/well and incubated at 37° C.overnight. Vaccinia virus engineered to express β-galactosidase andhemagglutinin genes (Vaccinia/HA) was added to the culture medium atconcentrations of 0 moi, 0.1 moi, or 1.0 moi and incubated at 37° C. forat least 3 days. Up to 90% of the cells were lysed within 72 hours ofinjection at 1.0 moi.

Example 8

[0060] This example demonstrates the destruction of mammary epitheliumby transfection with Vaccinia/HA in vivo.

[0061] The mammary glands of 45-day old virgin rats were injectedthrough the teat with 1×10⁷ particles of Vaccinia/HA in 20 μl 0.2%trypan blue (tracking dye). Contralateral control glands were injectedwith 0.2% trypan blue. The glands were excised after 3 days, fixed inchloroform:methanol:acetic acid and stained in iron-hematoxylin.Branching structures of a whole-mounted mammary gland injected withtracking dye alone were visible up to the end buds and alveoli. Alsovisible as brown bodies were the mammary lymph nodes. Examination of awhole-mounted mammary gland of the same rat receiving Vaccinia/HA intrypan blue on the contralateral side revealed that only about 30% ofthe ducts remained. In addition, the lymph nodes were considerablyenlarged, denoting the mounting of an immune response to clear theVaccinia from the vicinity;

[0062] All publications, patents, and patent applications cited hereinare hereby incorporated by reference to the same extent as if eachindividual document were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein.

[0063] While this invention has been described with emphasis uponpreferred embodiments, it will be obvious to those of ordinary skill inthe art that the preferred embodiments may be varied. It is intendedthat the invention may be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

What is claimed is:
 1. A method of treating the ductal epithelium of an exocrine gland prophylactically for cancer, which method comprises contacting the ductal epithelium of the exocrine gland with an epithelium-destroying agent to destroy less than all of the ductal epithelium so as to inhibit the formation of cancer of ductal epithelial origin.
 2. The method of claim 1 , wherein said epithelium-destroying agent is brought into contact with the ductal epithelium by ductal cannulation.
 3. The method of claim 2 , wherein said exocrine gland is a mammary gland.
 4. The method of claim 3 , wherein up to about 90% of the ductal epithelium is destroyed.
 5. The method of claim 4 , wherein up to about 80% of the ductal epithelium is destroyed.
 6. The method of claim 5 , wherein up to about 70% of the ductal epithelium is destroyed.
 7. The method of claim 3 , wherein essentially only the stem cells of the ductal epithelium are destroyed.
 8. The method of claim 3 , wherein said epithelium-destroying agent is a vector comprising a thymidine kinase gene combined with ganciclovir.
 9. The method of claim 8 , wherein said ganciclovir, instead of being brought into contact with the ductal epithelium by ductal cannulation, is brought into contact with the ductal epithelium by systemic administration.
 10. The method of claim 8 , which additionally comprises contacting the ductal epithelium with a cytokine or hematopoietic growth factor.
 11. The method of claim 10 , wherein said hematopoietic growth factor is GM-CSF.
 12. The method of claim 11 , wherein said GM-CSF is brought into contact with the ductal epithelium by ductal, cannulation of GM-CSF or a vector comprising a gene encoding GM-CSF.
 13. The method of claim 12 , wherein said vector comprising a gene encoding GM-CSF is the same vector comprising the thymidine kinase gene.
 14. The method of claim 3 , wherein said epithelium-destroying agent is a vector comprising a HPRT gene combined with HAT nucleotide.
 15. The method of claim 14 , wherein said HAT nucleotide, instead of being brought into contact with the ductal epithelium by ductal cannulation, is brought into contact with the ductal epithelium by systemic administration.
 16. The method of claim 14 , which additionally comprises contacting the ductal epithelium with a cytokine or hematopoietic growth factor.
 17. The method of claim 16 , wherein said hematopoietic growth factor is GM-CSF.
 18. The method of claim 17 , wherein said GM-CSF is brought into contact with the ductal epithelium by ductal cannulation of GM-CSF or a vector comprising a gene encoding GM-CSF.
 19. The method of claim 18 , wherein said vector comprising a gene encoding GM-CSF is the same vector comprising the HPRT gene.
 20. The method of claim 3 , wherein said epithelium-destroying agent is a vector comprising a gene, which, upon transformation of a cell of the ductal epithelium and expression therein, induces apoptosis or death of the transformed cell.
 21. The method of claim 20 , wherein said gene is bclxs.
 22. The method of claim 20 , which additionally comprises contacting the ductal epithelium of the mammary gland with a cytokine or hematopoietic growth factor.
 23. The method of claim 22 , wherein said hematopoietic growth factor is GM-CSF.
 24. The method of claim 23 , wherein said GM-CSF is brought into contact with the ductal epithelium by ductal cannulation of GM-CSF or a vector comprising a gene encoding GM-CSF.
 25. The method of claim 24 , wherein said vector comprising a gene encoding GM-CSF is the same vector comprising the apoptosis-inducing gene.
 26. The method of claim 3 , wherein said epithelium-destroying agent is ethanol.
 27. The method of claim 26 , which additionally comprises contacting the ductal epithelium of the mammary gland with a cytokine or hematopoietic growth factor.
 28. The method of claim 27 , wherein said hematopoietic growth factor is GM-CSF.
 29. The method of claim 3 , wherein said epithelium-destroying agent is a cytplytic virus.
 30. The method of claim 29 , wherein said cytolytic virus is a Vaccinia virus.
 31. The method of claim 30 , which additionally comprises contacting the ductal epithelium of the mammary gland with a cytokine or hematopoietic growth factor.
 32. The method of claim 31 , wherein said hematopoietic growth factor is GM-CSF.
 33. A method of treating the ductal epithelium of a mammary gland prophylactically for cancer, which method comprises contacting, by ductal cannulation, the ductal epithelium of the mammary gland with an epithelium-destroying agent selected from the group consisting of a vector comprising a thymidine kinase gene combined with ganciclovir, a vector comprising a HPRT gene combined with HAT nucleotide, a cytolytic virus, and ethanol so as to inhibit the formation of cancer of ductal epithelial origin.
 34. The method of claim 33 , wherein said cytolytic virus is a Vaccinia virus.
 35. The method of claim 33 , which additionally comprises contacting the ductal epithelium with a cytokine or hematopoietic growth factor.
 36. The method of claim 35 , wherein said hematopoietic growth factor is GM-CSF.
 37. A method of treating the ductal epithelium of an exocrine gland prophylactically for a disease, other than cancer, which affects the ductal epithelium of the exocrine gland, which method comprises contacting, by ductal cannulation, the ductal epithelium of the exocrine gland with an epithelium-destroying agent so as to destroy cells of the ductal epithelium affected by the disease.
 38. A method of treating the ductal epithelium of a mammary gland therapeutically for cancer, which method comprises contacting, by ductal cannulation, the ductal epithelium of the mammary gland with an epithelium-destroying agent, which does not specifically target cancerous cells, so as to destroy cancerous and noncancerous cells of the ductal epithelium and inhibit the spread of cancer.
 39. The method of claim 38 , wherein said epithelium-destroying agent is selected from the group consisting of a vector comprising a thymidine kinase gene combined with ganciclovir, a vector comprising a HPRT gene combined with HAT nucleotide, a cytolytic virus, and ethanol.
 40. The method of claim 39 , wherein said cytolytic virus is a Vaccinia virus.
 41. The method of claim 39 , which additionally comprises contacting the ductal epithelium with a cytokine or hematopoietic growth factor.
 42. The method of claim 41 , wherein said hematopoietic growth factor is GM-CSF.
 43. A method of treating the ductal epithelium of an exocrine gland therapeutically for a disease, other than cancer, which affects the ductal epithelium of the exocrine gland, which method comprises contacting, by ductal cannulation, the ductal epithelium of the exocrine gland with an epithelium-destroying agent so as to destroy cells of the ductal epithelium affected by the disease.
 44. A method of treating the ductal epithelium of a mammary gland both therapeutically and prophylactically for cancer, which method comprises: (a) treating said mammary gland therapeutically with a method selected from the group consisting of surgical removal of the cancerous tissue, radiation therapy, and/or chemotherapy; and (b) contacting, either concomitantly with or subsequently to (a), the ductal epithelium of the mammary gland with an epithelium-destroying agent, which does not specifically target cancerous cells, so as to destroy any remaining cancerous cells as well as noncancerous cells and inhibit the spread of cancer.
 45. The method of claim 44 , wherein said epithelium-destroying agent is selected from the group consisting of a vector comprising a thymidine kinase gene combined with ganciclovir, a vector comprising a HPRT gene combined with HAT nucleotide, a cytolytic virus, and ethanol.
 46. The method of claim 45 , wherein said cytolytic virus is a Vaccinia virus.
 47. The method of claim 45 , which additionally comprises contacting the ductal epithelium with a cytokine or hematopoietic growth factor.
 48. The method of claim 47 , wherein said hematopoietic growth factor is GM-CSF.
 49. The method of claim 44 , wherein said ductal epithelium is contacted by ductal cannulation. 